The present invention relates to a new agent for transferring nucleic acids into cells. This transfer agent is more particularly characterized in that it contains one or more disulphide bridges which are sensitive to reducing conditions. This new agent can be used to transfer nucleic acids of interest into different cell types either in vitro, in vivo or ex vivo.
With the development of biotechnology, the possibility of effectively transferring nucleic acids into cells has become a necessity. It involves the transfer of nucleic acids into cells in vitro, for example, for the production of recombinant proteins, or in the laboratory for studying the regulation of the expression of genes, the cloning of genes, or any other manipulation involving DNA. It may also involve the transfer of nucleic acids into cells in vivo, for example for the creation of transgenic animals, the production of vaccines, labelling studies or also therapeutic approaches. It may also be the transfer of nucleic acids into cells ex vivo, in approaches including bone marrow transplants, immunotherapy or other methods involving the transfer of genes into cells collected from an organism for the purpose of their subsequent readministration.
The various synthetic vectors developed so far in order to improve the transfer of nucleic acids into cells possess a considerable structural diversity which reflects the observation of the fact that their efficiency is different depending on the desired application and the intended cell types. This efficiency is largely dependent on their structure.
Among the synthetic vectors developed hitherto, cationic lipids have an important place. These vectors consist of a cationic polar part which interacts with the nucleic acids, and a hydrophobic lipid part which enables the complex formed to be protected from the external medium. The following may be mentioned by way of example: the monocationic lipids (DOTMA: Lipofectin(copyright)); lipopolyamines, in particular dioctadecylamidoglycyl spermine (DOGS) or 5-carboxy-spermylamide of palmitoylphosphatidylethanolamine (DPPES), whose preparation has been described, for example, in Patent Application EP 394 111; or else the cationic lipids cited in Applications WO 96/17823 and WO 97/18185 (incorporated into the present by way of reference).
Many studies have clearly indicated that cationic lipids possess properties which make it possible to promote transfection. However, it now appears necessary to develop cationic lipids having novel structures which make it possible to provide additional beneficial properties. Thus, there is a need for cationic lipids which would be more particularly suitable for crossing membrane barriers. Indeed, numerous obstacles prevent a real transfection efficiency, among which the difficulty for the nucleic acid to cross biological membranes and to penetrate into the cellular compartments (xe2x80x9cCellular and Molecular Barriers to Gene Transfer by a Cationic Lipidxe2x80x9d, Zabner, J. et al., J. Biol. Chem., 1995, No. 32, 18997-19007). It is this technical difficulty which the present invention proposes to solve.
Thus, the present invention relates to novel nucleic acid transfer agents which comprise at least one cationic hydrophilic region capable of noncovalently combining with nucleic acids and at least one lipophilic region, these regions being connected to each other through a so-called xe2x80x9cspacerxe2x80x9d arm, and comprising, in addition, at least one disulphide bridge positioned such that its reduction causes partial degradation of the lipophilic region, or alternatively positioned such that its reduction causes separation of the said transfer agent when it is symmetrical.
These transfer agents are capable of efficiently complexing nucleic acids by virtue of their cationic hydrophilic parts, this interaction strongly compacting the said nucleic acid, and the lipophilic region makes this ionic interaction insensitive to the external medium by covering the particle formed with a lipid film.
However, in addition to these properties which are desired for vectorization, the transfer agents according to the invention possess an extremely advantageous detergent property, by generating, at the level of the reducing cellular medium, because of the presence of the disulphide bridge(s), molecules of the polyaminated alkyl chain type which are membrane destabilizers. Indeed, the disulphide bridges are capable of constituting stable covalent bonds in oxidizing medium, and of breaking in reducing medium, according to the following scheme:
Xxe2x80x94Sxe2x80x94Sxe2x80x94Yxe2x86x92Xxe2x80x94SH+HSxe2x80x94Y
This type of structure is present, for example, in certain proteins possessing cysteine amino acids, and contributes to their three-dimensional structure and therefore to their biological activity. Disulphide bridges have, moreover, already been introduced into certain chimeric proteins, and in particular into immunotoxins, in order to connect the targeting domain to the active domain.
xe2x80x9cReducing mediumxe2x80x9d is understood to mean, for the purposes of the invention, a natural reducing medium, for example the intracellular medium, in particular the cytoplasm and in particular the endosomes. An artificial reducing medium representative of natural conditions is for example a medium comprising 0.1% to 20% of dithiotreitol (DTT).
By contrast, xe2x80x9coxidizing mediumxe2x80x9d is understood to mean any medium which is in contact with atmospheric oxygen and which contains no reducing agent, in particular the extracellular medium. A representative oxidizing medium for example consists of a 150 mM isotonic solution of sodium chloride, or of a solution containing 5% glucose.
The Applicant has thus demonstrated, quite unexpectedly, that one of the disulphide bridges could be introduced into a synthetic vector for the transfer of nucleic acids, in particular of the cationic lipid type, and that this did not affect its capacity to complex the nucleic acids in a non-reducing medium. It also shows that the nucleic acid transfer properties of these agents are preserved, or even improved. Moreover, the complexes formed are degraded in reducing medium, and therefore in particular in the cell, which makes it possible to generate detergent molecules, thus making a larger quantity of nucleic acid accessible to the cellular transcription machinery.
xe2x80x9cDetergentxe2x80x9d is understood to mean, for the purposes of the invention, any amphiphilic molecule having the property of being inserted into biological membranes and destabilizing them. This results from the capacity of detergents amphiphilic molecules to rupture the membranes by becoming inserted into the phospholipid double layers and by solubilizing the lipids and the proteins (La Cellule, Ed. Vigot and Dxc3xa9carie, 1988, pp. 581-583).
Another advantage of the transfer agents according to the invention also consists in their reduced intrinsic toxicity. Indeed, the transfer agent being degraded in the cell at the level of the disulphide bridges which are sensitive to reducing conditions, it does not exert the toxic effect observed for conventional transfer vectors. Furthermore, the improvement of the passage across the membranes allows the use of smaller doses of nucleic acid/transfer agent complex, with the beneficial consequences resulting therefrom on toxicity.
Finally, the Applicant has also demonstrated that the transfer properties are significantly improved when the lipophilicity of the transfer agents is sufficient and when they are used in adequate quantity. More particularly, it has been shown that one of the major advantages of increasing the lipophilicity of these agents, or of introducing a chain derived from a steroid, is the induction of improved resistance to serum.
The transfer agents according to the invention can have two types of structure, without this having an influence on their technical effect. In the first case, this structure can be represented in the following manner:
cationic hydrophilic region-spacer-lipophilic region
In such a structure, the disulphide bridge(s) are positioned in the lipophilic region so as to generate a detergent amphiphilic molecule when they are reduced.
The second type of structure can be represented as follows: 
In this case, the disulphide bridge(s) are positioned so that their reduction causes separation of the two symmetrical parts of the transfer agent, that is to say between the two spacer parts.
For the purposes of the invention, xe2x80x9ccationic hydrophilic partxe2x80x9d is understood to mean any hydrophilic molecule whose overall charge is positive at physiological pH, that is to say between pH 5 and 8, and possessing, in addition, properties of bonding with nucleic acids. This bond is in particular of the noncovalent bond type, such as for example ionic interactions. Preferably, the cationic hydrophilic region present in the transfer agents according to the invention is a polyamine or a polyaminoguanidine.
According to an advantageous variant, the cationic hydrophilic region corresponds to the following general formula: 
in which m is an integer greater than or equal to 2 and 1 is an integer greater than or equal to 1, it being possible for m to vary between the different groups of carbon between two amines. Preferably, m is between 2 and 6 inclusive, and 1 is between 1 and 5 inclusive. Still more preferably, the polyamine region is represented by spermine.
Another preferred polyamine region corresponds to the following general formula described in Application WO 97/18185: 
in which R1, R2 and R3 represent, independently of each other, a hydrogen atom or a group xe2x80x94(CH2)qxe2x80x94NRRxe2x80x2 with q being capable of varying between 1, 2, 3, 4, 5 and 6 independently between the different groups R1, R2 and R3, and R and Rxe2x80x2 represent, independently of each other, a hydrogen atom or a group xe2x80x94(CH2)qxe2x80x94NH2 with q defined as above, and m and n represent, independently of each other, an integer capable of varying between 0 and 6 with, when n is greater than 1, m being capable of taking different values and R3 different meanings in the above general formula.
The lipophilic region present in the transfer agents according to the invention consists of at least one fatty aliphatic chain and of one or more other aliphatic chains, of one or more steroid derivatives, of a natural or synthetic lipid, or optionally a combination of these, preferably capable of forming lamellar or hexagonal phases. These structures are characterized by the distances between the lamellae or the tubes which depend on the length of the fatty aliphatic chain or of the polar part of the lipid.
The term xe2x80x9cfatty aliphatic chainxe2x80x9d designates, for the purposes of the invention, a linear or branched alkyl chain comprising 10 to 22 carbon atoms, optionally saturated and/or fluorinated. Preferably, it comprises 12 to 22 carbon atoms. There may be mentioned more particularly the C12, C13, C14, C16, C18 and C19 aliphatic groups and the like, and in particular the (CH2)11CH3, (CH2)12CH3 (CH2)13CH3, and (CH2)17CH3 groups.
When the lipophilic region comprises a derivative of a steroid, the latter is preferably chosen from cholesterol, cholic acid or cholesterylamine.
In a preferred embodiment, the lipophilic region is composed of at least two fatty aliphatic chains. Still more preferably, it is composed of two or three fatty aliphatic chains.
According to another advantageous variant of the invention, the lipophilic part consists of a fatty aliphatic chain and a steroid derivative.
When the transfer agent according to the invention has a symmetrical structure, each symmetrical part of the molecule contains at least one fatty aliphatic chain.
The cationic hydrophilic region and the lipophilic region may be connected to each other through a so-called xe2x80x9cspacerxe2x80x9d arm. The spacer can be described, with no limitation being implied, as any acid or amine group comprising hydrolyzable functions, which is known to persons skilled in the art. Preferably, the so-called spacer region comprises an aliphatic or aromatic chain. Preferably, the spacer region may be, for example, chosen from amide, carbamate, ester or ether groups, or aromatic rings.
The transfer agents according to the invention comprise one or more disulphide bridges. The number of these bridges is determined by persons skilled in the art according to the structure of the transfer agent and the desired properties. Advantageously, the transfer agent comprises one or two disulphide bridges, and preferably one disulphide bridge. In the transfer agent, the disulphide bridge(s) may be positioned at different sites. The position depends on the number of bridges and the structure of the agent.
According to a first embodiment, the disulphide bridge is positioned such that its reduction causes partial degradation of the lipophilic region, thus generating a detergent amphiphilic molecule at the level of the cell. This partial degradation may correspond in particular to the loss of an aliphatic chain when the lipophilic region comprises several thereof, or alternatively the loss of the chain derived from a steroid when the lipophilic region contains one thereof. xe2x80x9cLoss of an aliphatic fatty chainxe2x80x9d is understood to mean either the complete loss thereof, or a partial loss, the remaining part,then being too short to constitute a fatty chain (length of less than 10 carbon atoms). Such a rupture destroys the integrity of the complex which then gradually disintegrates to give dissociated components of which at least one is a detergent amphiphilic molecule. The degradation of the complex can be easily checked by microscopy or by electrophoresis.
According to another variant, the disulphide bridge is positioned between the two spacer arms of a transfer agent of symmetrical structure, such that its reduction causes the separation of the said transfer agent.
According to another variant, the disulphide bridge is positioned between the two spacer arms of a transfer agent of symmetrical structure, such that its reduction causes the separation of the said transfer agent.
Preferred transfer agents according to the invention comprise:
as cationic hydrophilic region, a polyamine or polyaminoguanidine,
as lipophilic region, at least two fatty aliphatic chains, or at least one chain derived from a steroid and one fatty aliphatic chain, and,
a disulphide bridge whose reduction leads to the loss of a fatty aliphatic chain, or of a chain derived from a steroid when the transfer agent contains one.
In a particularly advantageous manner, the transfer agents of the invention comprise a polyamine region, three aliphatic chains of which at least two are fatty chains, and a disulphide bridge leading, in a reducing medium, to the loss of an aliphatic chain.
Other particularly advantageous transfer agents comprise a polyamine region, a fatty aliphatic chain and a chain derived from a steroid, and a disulphide bridge leading, in a reducing medium, to the loss of the chain derived from a steroid.
Other particularly advantageous transfer agents consist of two symmetric lipopolyamines, and of a disulphide bridge leading, in a reducing medium, to their separation.
Such agents are illustrated in the examples. The following compounds may be mentioned with no limitation being implied: 
Another subject of the invention relates to a composition comprising a transfer agent as defined above, and at least one nucleic acid. Preferably, the transfer agent and the nucleic acid are present in quantities such that the ratio of the positive charges of the agent to the negative charges of the nucleic acid is between 0.1 and 50. This ratio can be easily adjusted by persons skilled in the art depending on the agent used, the nucleic. acid and the type of cells to be transfected. Advantageously, this ratio is between 3 and 12 nanomoles of agent according to the invention per xcexcg of nucleic acid, and preferably between 3 and 9 nanomoles of transfecting agent per xcexcg of nucleic acid.
For the purposes of the invention, xe2x80x9cnucleic acidxe2x80x9d is understood to mean both a deoxyribonucleic acid and a ribonucleic acid. They may be natural or artificial sequences, and in particular genomic DNA (gDNA), complementary DNA (cDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), hybrid sequences or synthetic or semisynthetic sequences, oligonucleotides which are modified or otherwise. These nucleic acids may be of human, animal, plant, bacterial or viral origin and the like. They may be obtained by any technique known to persons skilled in the art, and in particular by the screening of libraries, by chemical synthesis or by mixed methods including the chemical or enzymatic modification of sequences obtained by the screening of libraries. They may be chemically modified, that is to say that they may be pseudonucleic acids (PNA), oligonucleotides modified by various chemical bonds (for example phosphorothioate or methyl phosphonate), or alternatively oligonucleotides which are functionalized, that is to say which are coupled with one or more molecules having distinct characteristic properties.
As regards more particularly deoxyribonucleic acids, they may be single- or double-stranded, as well as short oligonucleotides or longer sequences. In particular, the nucleic acids advantageously consist of plasmids, vectors, episomes, expression cassettes and the like. These deoxyribonucleic acids may carry genes of therapeutic interest, sequences for regulating transcription or replication, anti-sense sequences which are modified or otherwise, regions for binding to other cellular components, and the like.
Preferably, the nucleic acid comprises an expression cassette consisting of one or more genes of therapeutic interest under the control of one or more promoters and of a transcriptional terminator which are active in the target cells.
For the purposes of the invention, xe2x80x9cgene of therapeutic interestxe2x80x9d is understood to mean in particular any gene encoding a protein product having a therapeutic effect. The protein product thus encoded may be a protein, a peptide, and the like. This protein product may be homologous in relation to the target cell (that is to say a product which is normally expressed in the target cell when the latter has no pathological condition). In this case, the expression of a protein makes it possible, for example, to palliate an insufficient expression in the cell or the expression of a protein which is inactive or weakly active because of a modification, or to overexpress the said protein. The therapeutic gene may also encode a mutant of a cellular protein, having increased stability, a modified activity and the like. The protein product may also be heterologous in relation to the target cell. In this case, an expressed protein may, for example, supplement or provide an activity which is deficient in the cell, allowing it to combat a pathological condition, or to stimulate an immune response.
Among the products of therapeutic interest for the purposes of the present invention, there may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines [interleukins, interferons, TNF, and the like (FR 9,203,120)], growth factors, neurotransmitters or their precursors or synthesis enzymes, trophic factors [BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP/pleiotrophin, and the like], dystrophin or a minidystrophin (FR 91/11947), the CFTR protein associated with cystic fibrosis, tumour suppressor genes [p53, Rb, Rap1A, DCC, k-rev, and the like (FR 93/04745)], genes encoding factors involved in coagulation [factors VII, VIII, IX], the genes involved in DNA repair, suicide genes [thymidine kinase, cytosine deaminase], the genes for haemoglobin or other protein carriers, the genes corresponding to the proteins involved in the metabolism of lipids, of the apolipoprotein type chosen from apolipoproteins A-I, A-II, A-IV, B, C-I, C-II, C-III, D, E, F, G, H, J and apo(a), metabolic enzymes such as for example lipoprotein lipase, hepatic lipase, lecithin cholesterol acyl transferase, 7-alpha-cholesterol hydroxylase, phosphatidic acid phosphatase, or lipid transfer proteins such as the cholesterol ester transfer protein and the phospholipid transfer protein, an HDL-binding protein or a receptor chosen, for example, from the LDL receptors, the remnant chylomicron receptors and the scavenger receptors, and the like.
The therapeutic nucleic acid may also be a gene or an anti-sense sequence, whose expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such sequences can, for example, be transcribed in the target cell into RNAs which are complementary to cellular mRNAs and thus block their translation to protein, according to the technique described in Patent EP 140 308. The therapeutic genes also comprise the sequences encoding ribozymes, which are capable of selectively destroying target RNAs (EP 321 201).
As indicated above, the nucleic acid may also comprise one or more genes encoding an antigenic peptide, which is capable of generating an immune response in humans or in animals. In this specific embodiment, the invention therefore allows the production of vaccines or the carrying out of immunotherapeutic treatments applied to humans or to animals, in particular against microorganisms, viruses or cancers. They may be in particular antigenic peptides specific for the Epstein-Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, the syncitia forming virus, other viruses, or specific for tumours (EP 259 212).
Preferably, the nucleic acid also comprises sequences allowing the expression of the therapeutic gene and/or the gene encoding the antigenic peptide in the desired cell or organ. They may be sequences which are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the infected cell. They may also be sequences of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences derived from the genome of the cell which it is desired to infect. Likewise, they may be promoter sequences derived from the genome of a virus. In this regard, there may be mentioned, for example, the promoters of the E1A, MLP, CMV and RSV genes, and the like. In addition, these expression sequences may be modified by the addition of activating or regulatory sequences, and the like. The promoter may also be inducible or repressible.
Moreover, the nucleic acid may also comprise, in particular upstream of the therapeutic gene, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell. This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence. The nucleic acid may also comprise a signal sequence directing the synthesized therapeutic product towards a particular compartment of the cell.
The compositions may, in addition, comprise adjuvants capable of combining with the transfer agent/nucleic acid complex and of improving its transfecting power.
In this regard, the compositions according to the invention may comprise, as adjuvant, one or more neutral lipids. Such compositions are particularly advantageous, in particular when the ratio of the positive charges of the agent to the negative charges of the nucleic acid is low. The Applicant has indeed shown that the addition of a neutral lipid makes it possible to improve the formation of nucleolipid particles and, surprisingly, to promote cellular penetration by destabilizing the membrane.
More preferably, the neutral lipids used within the framework of the present invention are lipids containing two fatty chains.
In a particularly advantageous manner, natural or synthetic lipids which are zwitterionic or lacking ionic charge under physiological conditions are used. They may be chosen more particularly from dioleoyl-phosphatidylethanolamine (DOPE), oleoylpalmitoyl-phosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -cholesteryl, -myristoylphosphatidylethanolamines as well as their derivatives which are N-methylated one to three times, phosphatidyl glycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins), or asialogangliosides (such as in particular asialoGM1 and GM2). These different lipids may be obtained either by synthesis or by extraction from organs (for example the brain) or from eggs, by conventional techniques well known to persons skilled in the art. In particular, the extraction of the natural lipids may be carried out by means of organic solvents (see also Lehninger Biochemistry).
More recently, the Applicant has demonstrated that it was also particularly advantageous to use, as adjuvant, a compound directly involved or otherwise in the condensation of the said nucleic acid, such as those cited in Application WO 96/25508. The presence of such a compound in a lipopolyamine-based transfecting composition makes it possible to considerably reduce the quantity of this agent, with the beneficial consequences resulting therefrom from the toxicological point of view, without any damaging effect on the transfecting activity of the said composition. xe2x80x9cCompound involved in the condensation of the nucleic acidxe2x80x9d is intended to define a compound which compacts, directly or otherwise, the nucleic acid. More precisely, this compound may either act directly at the level of the nucleic acid to be transfected, or may be involved at the level of an additional compound which is directly involved in the condensation of this nucleic acid. Preferably, it acts directly at the level of the nucleic acid. In particular, the precompacting agent may be any polycation, for example polylysine. According to a preferred embodiment, this agent which is involved in the condensation of the nucleic acid is derived, as a whole or in part, from a protamine, a histone, a nucleolin and/or one of their derivatives. Such an agent may also consist, as a whole or in part, of peptide units (KTPKKAKKP) and/or (ATPAKKAA), it being possible for the number of units to vary between 2 and 10. In the structure of the compound according to the invention, these units may be repeated continuously or otherwise. They may thus be separated by linkages of a biochemical nature, for example one or more amino acids, or of a chemical nature.
Preferably, the compositions of the invention comprise from 0.01 to 20 equivalents of adjuvant per equivalent of nucleic acids in weight/weight, and more preferably from 0.5 to 5.
The compositions according to the invention may also involve one or more targeting elements which make it possible to direct the nucleic complexes towards receptors or ligands at the surface of the cell. By way of example, the composition of the present invention may comprise one or more antibodies directed against cell surface molecules, or one or more membrane receptor ligands such as insulin, transferrin, folic acid or any other growth factor, cytokines or vitamins. Advantageously, the composition may use lectins, modified or otherwise, in order to target particular polysaccharides at the surface of the cell or on the neighbouring extracellular matrix. Proteins with an RGD unit, peptides containing a tandem of RGD units, which is cyclic or otherwise, as well as polylysine peptides, can be used. More recently, natural or synthetic ligand peptides have also been described which are advantageous in particular for their selectivity towards specific cells and which are capable of efficiently promoting internalization in these cells (Bary et al., Nature Medicine, 2, 1996, 299-305). These targeting agents are generally conjugated to the cationic transfection agent considered.
The invention also extends to any composition as defined above and comprising, in addition, one or more other agents known for transfecting the nucleic acid. In particular, there may be mentioned the products described in the Patent EP 394 111 and in Patent Applications WO 96/17823, or WO 97/18185 (incorporated into the present by way of reference).
Another subject of the present invention also relates to the use of a transfer agent as defined above for transferring nucleic acids into cells.
The compositions comprising the transfer agent according to the invention can be formulated for administration by the topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal or intraperitoneal route, and the like. Preferably, the pharmaceutical compositions of the invention contain a vehicle which is pharmaceutically acceptable for an injectable formulation, in particular a direct injection into the desired organ, or for administration by the topical route (on the skin and/or the mucous membrane). They may be in particular isotonic sterile solutions, or dry, in particular freeze-dried, compositions which, upon addition, depending on the case, of sterilized water or of physiological saline, allow the constitution of injectable solutions. The nucleic acid doses used for the injection as well as the number of administrations may be adapted according to various parameters, and in particular according to the mode of administration used, the relevant pathological condition, the gene to be expressed, or the desired duration of treatment. As regards more particularly the mode of administration, it may be either a direct injection into the tissues or the circulatory system, or a treatment of cells in culture followed by their reimplantation by injection or transplantation.
The invention relates, in addition, to a method of transferring nucleic acids into cells comprising the following steps:
(1) bringing the nucleic acid into contact with a transfer agent as defined above., to form a nucleic acid/transfer agent complex,
(2) bringing the cells into contact with the complex formed in (1).
In the case of a composition comprising one or more other transfection agents and/or one or more adjuvants, step (1) is preceded by a step of bringing the different transfection agents into contact and/or by a step of bringing the transfection agent into contact with the adjuvant(s)
The agent according to the invention/nucleic acid complexes are formed by mixing, volume for volume, two solutions, one containing the transfection agent according to the invention in the form of micelles or of a hexagonal lamellar phase, the other the nucleic acid to be transfected. The complexes are formed within a few seconds. They may be negatively or positively charged or they may be neutral, depending on the quantity of lipid added to the nucleic acid (Pitard B. et al., Proc. Natl. Acad. Sci. USA, Vol. 94, pp. 14412-14417, December 1997). The sizes of these complexes vary between 50 and 300 nm in diameter (measured by a quasielastic diffusion of light and by transmission electron microscopy). Moreover, the morphology of the complexes varies with the charge ratio R (ratio of the positive charges provided by the cationic lipid to the negative charges provided by the nucleic acid). For example, the negatively charged complexes are surrounded by molecules of nucleic acid. Moreover, the positively charged complexes have cationic lipids at their surface. As for the neutral complexes, they are. colloidally unstable. The Applicant has thus shown that it was possible to stabilize them by adding a non-ionic surfactant in a sufficient quantity. Preferred surfactants are in particular poloxamers, polyoxyethylene alcohols, polyoxyethylene nonyl phenyl ether or PEGs (polyethylene glycols) with a dendritic benzyl polyether head.
The cells are brought into contact with the complex by incubating the. cells. with the said complex (for uses in vitro or ex vivo), or by injecting the complex into an organism (for uses in vivo). The incubation is carried out preferably in the presence, for example, of 0.01 to 1000 xcexcg of. nucleic acid per 106 cells. For in vivo administration, doses of nucleic acids ranging from 0.01 to 10 mg may be used.
The transfer agents according to the invention are particularly advantageous for their use in transferring nucleic acids into primary cells or into established lines. These may be friboblast cells, muscle cells, nerve cells (neurons, astrocytes, glial cells), hepatic cells, haematopoietic cells (lymphocytes, CD34, dendritic cells, and the like), epithelial cells and the like in diffentiated or pluripotent form (precursors).
The present invention thus provides a particularly advantageous method for the treatment of diseases comprising the in vivo, ex vivo or in vitro administration of a nucleic acid capable of correcting the said disease, combined with a compound according to the invention. More particularly, this method is applicable to diseases resulting from a deficiency in or a lack of protein or nucleic product. The administered nucleic acid encodes the said protein product or contains the said nucleic product.
In addition to the preceding arrangements, the present invention also comprises other characteristics and advantages which will emerge from the examples and figures which follow, and which should be considered as illustrating the invention without limiting the scope thereof. In particular, the Applicant proposes, with no limitation being implied, various operating protocols as well as reaction intermediates which can be used to prepare the transfer agents according to the invention. of course, it is within the capability of persons skilled in the art to draw inspiration from these protocols or intermediate products in order to develop similar methods so as to lead to these same compounds.